In the last grant period, our studies focused on intrinsic determinants of neuronal vulnerability in Parkinson's disease (PD). In the renewal application, we propose to pursue extrinsic, network-based determinants of neuronal vulnerability in PD, focusing on the innervation the innervation of the SNc by the pedunculopontine nucleus (PPN). The PPN provides the SNc with a rich glutamatergic and cholinergic innervation. Both glutamatergic and cholinergic signaling have been implicated in PD pathogenesis, but until recently, rigorous characterization of the role of these two PPN projection systems in regulating the activity and stress levels of SNc dopaminergic neurons has not been possible. This situation has now fundamentally changed with the advent of optogenetic and pharmacogenomic approaches. Using these new tools, we propose to pursue four specific aims with translational potential. Specific Aim 1: Determine whether PPN glutamatergic synapses on SNc dopaminergic neurons increase their oxidant stress. Our working hypothesis is that PPN terminal release of glutamate activates GluN2D-containing NMDA receptors (NMDARs) in proximal dendrites of SNc dopaminergic neurons, leading to an elevation in Ca2+ concentration and mitochondrial oxidant stress. Specific Aim 2: Determine whether PPN cholinergic synapses on SNc dopaminergic neurons decrease their oxidant stress. Our working hypothesis is that PPN terminal acetylcholine release activates 42- and 62-containing postsynaptic nicotinic receptors (nAChRs) and that repetitive activation of these terminals, leads to suppression in Cav1.3 channel currents and diminished mitochondrial oxidant stress. Specific Aim 3: Determine whether chronic nicotine administration decreases mitochondrial oxidant stress in SNc DA neurons. Our working hypothesis is that chronic nicotine administration suppresses intracellular Ca2+ oscillations driven by Cav1.3 channels during pacemaking and dis-facilitates glutamate release from PPN glutamatergic terminals, leading to a significant drop in mitochondrial oxidant stress and enhanced function. Specific Aim 4: Determine whether pharmacogenomic suppression of PPN glutamatergic neurons enhances the survival and function of SNc dopaminergic neurons in a mouse model of PD. Our working hypothesis is that dopamine depletion drives pathophysiological activity in PPN glutamatergic neurons, which increases oxidant stress levels in SNc DA neurons and accelerates their loss in PD models. The achievement of these aims could lead to the development of novel drugs targeting specific sub-classes of NMDARs and nAChRs that could be used in combination with Cav1.3 Ca2+ channel antagonists to slow or stop the progression of PD.